Retrospective Analysis Of Patients With BK Virus-associated Nephropathy And Acute T-cell Mediated Rejection After Renal Transplantation And A Microarray Analysis Of Serum Cytokines

Part ?.Retrospective analysis of patients with BK virus-associated nephropathy and T cell-mediated acute rejection after renal transplantation and establishment of a differential diagnosis modelBackground and purpose:With the use of new immunosuppressive agents,the infection rate of BK virus becomes higher,and the BK virus-associated nephropathy(BKN)has become one of the important causes for graft failure.Its clinical and pathological findings need to be differentiated from T cell-mediated acute rejection(TCMR).At present,there are few reports on the comparative analysis of the clinical features of BKN and TCMR.SV40 large T-antigen staining method,which is gold standard for BKN diagnosis,has certain limitations.Therefore,in this retrospective analysis,we summarize and compare the clinical pathological features and prognostic factors of BKN and TCMR cases,and establish a diagnostic model to better distinguish BKN from TCMR.METHODS:We analyzed data from the First Hospital Affiliated to Zhejiang University during the period from 2011.1 to 2018.3.The renal biopsy confirmed 54 patients with BKN and 237 patients with TCMR.The clinical baseline data of the patients,inflammatory cell infiltration of pathological tissues,laboratory examination and data in follow-up period were collected.The survival curves of BKN group and TCMR group were drawn by Kaplan.Meier method and multi-factor COX regression model.The prognosis of the two groups was compared.Independent risk factors affecting the cumulative survival rate of graft in the BKN group and the TCMR group were screened by multivariate COX regression model.Finally,the binary logistic regression model was used to establish the differential diagnosis model between BKN group and TCMR group.All the variables were included in the model,and the independent predictors supporting BKN diagnosis were selected.Finally,a new diagnostic model was established and the ROC curve was drawn as well.RESULTS:A total of 54 patients with BKN(mean age 40.2±10.6 y,40.7%female)and 237 patients with TCMR(mean age 40.1±12.1 y,female 32.9%)were included in the study.In the baseline,the number of urine red blood cells in the BKN group was significantly lower than those in the TCMR group[4.9(2,10.25)vs 8.2(3.8,22.6),P=0.0066)](/p L),and the urine protein of the BKN group was lighter than the TCMR group[0(0,0.2)VS 0.2(0.045,0.49),P<0.0001](g/L).The levels of hemoglobin and serum albumin in the BKN group were significantly higher than those in the TCMR group[HB(g/L):120.4± 19.6 VS 110.8± 22.6,P=0.0042][Albumin(g/L):45.0±7.0 VS 41.3±7.1,P<0.0001].The total cholesterol level in the BKN group was higher than those in the TCMR group(4.6± 1.0 VS 4.3±1.0,P=0.022)(mmol/L).The proportion of IL-2 receptor antagonist or ATG use in the BKN group was significantly higher than the TCMR group(P=0.0021).The baseline tacrolimus concentration in the BKN group was significantly higher than the TCMR group(8.6±6.9 vs 6.0±2.7,P=0.0014).The infiltration of a-SMA positive cells,CD3 positive cells,and CD68 positive cells in the BKN group was significantly severer than the TCMR group(P>0.05)By comparing renal function of BKN group and TCMR group during follow-up period,it was indicated that the overall renal function of the BKN group was worse than that of the TCMR group.Kaplan.Meier survival curve showed that patients with BKN seemed to have worse graft survival rate compared with TCMR group.The median survival time of the transplanted kidney in the TCMR group was 2166(1767.7,2564.3)days,and the median survival time of the BKN group was 1800(898.3,2701.6)days(Log Rank test P=0.06).After adjusting the time after kidney transplantation,baseline eGFR,serum albumin,PRA and other factors by multivariate COX regression analysis model,we found that BKN group had a significantly higher graft failure rate than TCMR group(P<0.001).Based on multivariate COX regression analysis model,it was revealed that urine protein levels(OR=6.786,95%Cl:2.170-21.220,P=0.001),steroid therapy(OR=2.831,95%Cl):1.338-5.990,P=0.006),and CD3 positive cell infiltration of biopsy tissue(OR=5.610,95%CI:1.400-22.482)were independent risk factors for cumulative renal allograft survival rate in patients with BKN.PRA-I antibody levels(OR=3.972,95%CI:1.440-10.956,P=0.008),low serum albumin(OR=2.146,95%CI:1.011-4.557,P=0.047)and baseline renal function CKD grading(OR=2.713,95%CI:1.803-4.081,P<0.0001)were independent risk factors for cumulative renal allograft survival rate in patients with TCMR.Finally,we used binary logistic regression model and ROC curve to establish a differential diagnosis model of BKN and TCMR.The cut-off valure is 0.179,the sensitivity of this model is 96.3%,the specificity is 90.6%,and the prediction accuracy is 96.1%..The expression of this formula is:P(Y=1|X)=1/1+e6.617-1.034*X1+1.552*X2-0.681*X3 where(X1 is the degree of infiltration of CD20 positive cells in pathological tissue,X2 is the urinary protein level at the time of diagnosis,and X3 represents l0log urine BK virus DNA value)CONCLUSION:BKN patients have a lower level of proteinuria and urinary red blood cells compared to patients with TCMR.The inflammatory cells infiltration of the pathological tissue was similar between the two groups,but infiltration of a-SMA positive cells,CD3 positive cells,and CD68 positive cells in the BKN group was severer than that in the TCMR group.Renal function and graft survival were significantly worse in the BKN group during the follow-up period.Finally,we established a differential diagnosis model of BKN and TCMR.Part ? A microarray analysis of serum cytokines of patients with BK virus-associated nephropathy and T cell-mediated acute rej ectionBACKGROUND AND PURPOSE:The clinical and pathological findings of BK virus-associated nephropathy(BKN)and T cell-mediated acute rejection(TCMR)are similar.At present,there are some studies trying to characterize BKN and TCMR at a molecular level.There are reports comparing BKN and TCMR on a transcriptome levels by using peripheral blood and renal biopsy tissues,and a few reports focusing on small molecule metabolites and blood urine.We still don't know which role the cytokines has played in the pathogenic process of BKN and TCMR.This study intends to draw the cytokine maps of BKN and TCMR patients,screen differentially expressed cytokines.Thus,we can better understand and distinguish the process of BK and TCMR,and provide clues for the potential molecular markers in the future.METHODS:Eight patients with BKN,8 patients with TCMR and 8 patients with stable renal function were included during 2014.1.1 to 2016.1.1.All the patients underwent renal transplantation surgery at the First Affiliated Hospital of Zhejiang University,and regularly visited our outpatient clinic during the follow-up time.Serum samples of those patients were detected using the GSH-CAA-440 protein chip supplied by RayBiotech.RESULTS:In this study,high-throughput cytokine microarrays were used to screen for different cytokines in 8 BKN patients,8 TCMR patients,and 8 creatinine-stable renal transplant recipients.There were no significant differences in baseline data between three groups.The study found that there were 29 cytokines different expressed in the BKN group compared with TCMR group,6 proteins up-regulated(ANG-4,CXCL-14,etc.)and 23 proteins down-regulated(IL-2 Ra,IL-23,IL-21,etc.).These proteins are relevant to the cytokine signal recognition pathway,infection-related pathway,HIF-1 pathway,Th17 cell differentiation pathway,JNK-STAK pathway and IL17-related signaling pathway;Compared with stable renal function group,61 cytokines were different expressed in BKN,with 25 cytokine up-regulated,such as ANG-4,G-CSF R,etc.and with 36 cytokine down-regulated,such as BMPR-IB,Cadherin-13,etc.These proteins are involved in the PI3K-Akt pathway,MAPK pathway,JAK-STAT pathway,infection,RAS signaling pathway,etc.17 cytokines expressed differently in the TCMR group compared with the stable renal function group,with 15 cytokine up-regulated,such as NT-4,MMP-8,etc.and with two cytokines down-regulated,which are CXCL14 and Midkine.These proteins are involved in cytokine signal recognition pathway,IL-17 signaling pathway,TNF pathway,etc.CONCLUSION:High-throughput protein chip is an ideal technical method for screening BKN and TCMR serum cytokines.This result suggests that the PI3K-Akt pathway,MAPK pathway,JAK-STAT pathway,infection,RAS signaling pathway may contribute to the pathogenesis of BKN,while cytokine signaling pathway,IL-17 signaling pathway,TNF pathway may be associated with TCMR.The 29 different proteins between BKN group and TCMR group may be potential molecular markers for identification of BKN and TCMR.